A novel protective effect of erythropoietin in the infarcted heart

Abstract

Erythropoietin (EPO) has been shown to protect neurons from ischemic stroke, but can also increase thrombotic events and mortality rates in patients with ischemic heart disease. We reasoned that benefits of EPO might be offset by increases in hematocrit and evaluated the direct effects of EPO in the ischemic heart. We show that preconditioning with EPO protects H9c2 myoblasts in vitro and cardiomyocytes in vivo against ischemic injury. EPO treatment leads to significantly improved cardiac function following myocardial infarction. This protection is associated with mitigation of myocyte apoptosis, translating into more viable myocardium and less ventricular dysfunction. EPO-mediated myocyte survival appears to involve Akt activation. Importantly, cardioprotective effects of EPO were seen without an increase in hematocrit (eliminating oxygen delivery as an etiologic factor in myocyte survival and function), demonstrating that EPO can directly protect the ischemic and infarcted heart.

Figures

Figure 1

EPO-mediated activation of critical intracellular kinase cascades including cell survival pathways. Shown are representative Western blots for ERK (a), Akt (b), Jak1 (c), and STAT3 (d) over serial time points. Activated (p-) signaling intermediates were assessed using anti-phospho Ab’s, and activated forms were normalized to total protein content of the corresponding molecule (lower panel of all blots). Data are representative of n = 3–4 experiments.

Figure 2

Apoptotic cell death in H9c2 myoblasts exposed to oxidative stress and hypoxia. (a) The ratio of apoptotic cells to total adherent cells in the dish following oxidative stress (H2O2 exposure). Cells were treated with H2O2 (200 μM) following the treatment with EPO (0.4 or 10 U/ml, n = 8 each). Cells were stained with Hoechst 33258 dye, and nuclear morphology was revealed by fluorescent microscopy (described in Methods). Data shown are the mean ± SEM. *P < 0.05 versus untreated cells. (b) The ratio of apoptotic cells to total adherent cells in the dish after hypoxic injury. Cells were exposed to anoxia (12 hours) and percentage of nuclear fragmentation quantified under the following conditions: vehicle (control), DMSO (n = 4), white bars; wortmanin (n = 4), black bars; and PD98059 (n = 4), gray bars. Presence (8 U/ml) or absence of EPO is indicated by + and –, respectively. Cells were stained as above. *P < 0.05 versus vehicle-treated (DMSO), †P < 0.05 versus EPO alone. (c and d) Representative sample of H9c2 cells treated with H2O2 (200 μM) without EPO pretreatment (c) or with EPO (10 U/ml) for 24 hours (d). Arrows indicate fragmented nuclei under both conditions.

Figure 3

In vivo cardiac physiology in post-MI rabbits. Hemodynamic measurements at post-MI day 3 in the experimental groups MI + saline (n = 11), filled diamonds; MI + EPO (n = 12), filled squares; and normal sham rabbits (n = 5), filled triangles. Measurements were taken at baseline (0) (see Table 1) and after progressive ISO stimulation. Data is the mean ± SEM. *P < 0.05 versus MI + saline; †P < 0.05 versus sham (ANOVA). (a) Global cardiac function measured by LV dP/dtmax. (b) LV relaxation as measured by LV dP/dtmin. (c) Heart rate. (d) LVEDP (EDP).

Figure 4

Hematocrit values starting at day 0 (MI) and post-MI days 1, 3, and 4 in rabbits treated with EPO (MI + EPO, white bars, n = 12) or saline (MI + saline, black bars, n = 11). Data are the mean ± SEM. *P < 0.05 versus MI + saline.

Figure 5

LV area at risk quantification after ischemia/reperfusion. (a) Graphic representation of the LV infarction size measured as the percentage of infarct of total ischemic area at risk in MI + saline (control) rabbits (black bar, n = 5) and MI + EPO rabbits (white bar, n = 5). The LV area at risk was virtually identical in both groups (see Results). Data are the mean ± SEM. *P < 0.005 versus MI + saline. (b) Representative LV cross sections from a MI + saline (control) rabbit and (c) from a MI + EPO rabbit. The dark blue–stained areas are nonischemic tissue, and red-stained areas represent the ischemic LV area at risk. Infarcted areas are indicated by blanched (yellow-white) areas within this area at risk, with much less seen in the MI + EPO heart (c).

Figure 6

TUNEL staining and quantification of apoptosis in vivo in postischemic rabbit hearts. (a) Graphic representation of TUNEL-positive nuclei in MI + saline (control) rabbit hearts (black bar, n = 4) and MI + EPO rabbits (white bar, n = 4). Data shown are the mean ± SEM of the number of TUNEL-positive cells (nuclei) per high-power field analyzed by microscopy. *P < 0.05 versus MI + saline (control) rabbits. (b) Representative TUNEL-stained LV section from an MI + saline (control) rabbit showing several positive-stained apoptotic nuclei. (c) Representative TUNEL-stained LV section from a MI + EPO rabbit with very few apoptotic nuclei.

Figure 7

In vivo activation of signaling kinases in the intact adult rabbit heart after EPO treatment. Shown in graphic representation is the activation of (a) Akt and (b) ERK assessed in LV lysates prepared 12 hours after the administration of EPO (1,000 U/kg) or saline (Control). Activated Akt and ERK were assessed using anti-phospho Ab’s, and activated forms were normalized to total protein content of the corresponding molecule as in Figure 1. *P < 0.05 versus control values (n = 4 each).